Regulation of UDP-Galactose:Ceramide Galactosyltransferase and UDP-Glucose:Ceramide Glucosyltransferase After Crush and Transection Nerve Injury

The enzyme activities of ceramide galactosyltransferase and ceramide glucosyltransferase were assayed as a function of time (0, 1, 2, 4, 7, 14, 21, 28, and 35 days) after crush injury or permanent transection of the adult rat sciatic nerve. These experimental models of neuropathy are characterized by the presence and absence of axonal regeneration and subsequent myelin assembly. Within the first 4 days after both injuries, a 50% reduction of ceramide galactosyltransferase-specific activity was observed compared to values found in the normal adult nerve. This activity remained unchanged at 7 days after injury; however, by 14 days the ceramide galactosyltransferase activity diverged in the two models. The activity increased in the crushed nerve and reached control values by 21 days, whereas a further decrease was observed in the transected nerve such that the activity was nearly immeasurable by 35 days. In contrast, the ceramide glucosyltransferase activity showed a rapid increase between 1 and 4 days, followed by a plateau that was 3.4-fold greater than that in the normal adult nerve, which persisted throughout the observation period in both the crush and transection models. [3H]Galactose precursor incorporation studies at 7, 14, 21, and 35 days after injury confirmed the previously observed shift in biosynthesis from the galactocerebrosides during myelin assembly in the crush model to the glucocerebrosides and oligohexosylceramide homologues in the absence of myelin assembly in the transection model. The transected nerves were characterized by a peak of biosynthesis of the glucocerebrosides at 14 days. Of particular interest is the biosynthesis of the glucocerebrosides and the oligohexosylceramides at 7 and 14 days after crush injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Androgen receptor locus on the human X chromosome: regional localization to Xq11-12 and description of a DNA polymorphism

The gene for the androgen receptor, mutations at which cause the X-linked androgen insensitivity syndrome, has been localized to the q11----q12 region of the human X chromosome by analysis, using a cloned cDNA for the androgen receptor, of somatic cell hybrid panels segregating portions of the X chromosome. A moderate-frequency HindIII RFLP has been found which should be useful in genetic linkage analysis of the various inherited forms of androgen insensitivity.     

Structure/activity relationships in the arginine taste pathway of the channel catfish

To characterize the molecular/structural requirements for activationor antagonism of the arginine taste pathways in catfish, Ictaluruspunctatus, structure/activity studies were performed using integratedmultiunit responses and cross-adaptation. Of all the guanidinium-containingcompounds tested, only L-arginine, L--amino-ß-guanidinopropionic acid (L-AGPA) and L-arginine methyl and ethyl esterswere strong stimuli. Results of functional group substitutionsand modification of the L-arginine parent molecule indicatedthat: (i) stereospecificity was observed with D-arginine beinga much less effective stimulus than L-arginine; (ii) an L-aminogroup must be present and unblocked (-chloro-guanidino-N-valericacid and N-acetyl L-arginine were weak or inactive stimuli);(iii) a free carboxylic acid group was not necessary for activity;(iv) the distance between the anomeric carbon and the guanidiniumgroup was not critical (L-AGPA, having two methylene groupsless than L-arginine was a moderately strong stimulus as wasL-canavanine) and (v) modification or substitution of the guanidinumgroup by other basic groups including amine, methyl or dimethylamineor by an isosterc (ureido) resulted in loss of stimulatory ability.In general, those stimuli and analogs that were good cross-adaptersof L-arginine stimulation were also good competitors for L-[³H]argininebinding to a partial membrane fraction (P2) from catfish tasteepithelium. On the other hand, compounds that were poor cross-adaptingstimuli were also poor binding competitors. While D-argininewas a poor stimulus, it did cross-adapt L-arginine and competedwell with L-[³H]arginine for binding to fraction P2.     

Anticoagulant activity of synthetic hirudin peptides

Synthetic peptides based on the COOH-terminal 21 residues of hirudin were prepared in order to 1) evaluate the role of this segment in hirudin action toward thrombin, 2) define the shortest peptide derivative with anticoagulant activity, and 3) investigate the role of tyrosine sulfation in the peptides' inhibitory activities. A hirudin derivative of 20 amino acids, Hir45-64 (derived from residues 45-64 of the hirudin polypeptide), was found to effect a dose-dependent increase in the activated partial thromboplastin time (APTT) of normal human plasma but to have no measurable inhibitory activity toward thrombin cleavage of a tripeptidyl p-nitroanilide substrate. Anticoagulant activity in hirudin derivatives was comparable in peptides of 20, 16, and 12 residues truncated from the NH2 terminus. Additional truncated peptides prepared by synthesis and carboxypeptidase treatment reveal that the minimal sequence of a hirudin peptide fragment with maximal anticoagulant activity is contained within the sequence: NH2-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-COOH. The 12-residue derivative thus identified was reacted with dicyclohexylcarbodiimide in the presence of sulfuric acid to yield a Tyr-sulfated peptide, S-Hir53-64. By comparison to unsulfated peptide, S-Hir53-64 was found to contain a specific inhibitory activity enhanced by one order of magnitude toward increase in APTT and to effect a dose-dependent increase in thrombin time of normal human plasma to yield a 4-fold increase in thrombin time with 2.5 micrograms/ml peptide using 0.8 units/ml alpha-thrombin. Comparison of S-Hir53-64 to hirudin in thrombin time and APTT assays reveals a 50-fold difference in molar specific activities toward inhibition of thrombin. Comparison of antithrombin activities of S-Hir53-64 using a variety of animal thrombins demonstrates greatest inhibitory activity toward murine, rat, and human enzymes and a 10-fold reduced activity toward bovine thrombin.     

Reduction of 3-methoxytyramine concentrations in the caudate nucleus of rats after exposure to high-energy iron particles: evidence for deficits in dopaminergic neurons

Exposure to low doses of high-energy iron particles can alter motor behavior. The ability of rats to hang from a wire has been reported to be significantly degraded after exposure to doses as low as 0.5 Gy. In addition, deficits in the ability of acetylcholine to regulate dopamine release in the caudate nucleus (an area in the brain important for motor function) have been found. The concentrations of 3-methoxytyramine (3-MT), a metabolite of dopamine whose concentrations reflect dopamine release in vivo, were measured after rats were exposed to different doses of high-energy iron particles to gain further information about the effect of radiation on the dopaminergic system. Concentrations of 3-MT were significantly reduced 3 days after exposure to 5 Gy but returned to control values by 8 days. After 6 months, concentrations were again less than control values. Exposure to 5 Gy of high-energy electrons or gamma photons had no effect 3 days after exposure. Very high doses of electrons were needed to alter 3-MT concentrations. One hundred grays of electrons decreased 3-MT 30 min after irradiation but levels returned to control values by 60 min. Gamma photons had no effect after doses up to 200 Gy. These results provide further evidence that exposure to heavy particles can degrade motor behavior through an action on dopaminergic mechanisms and that this can occur after doses much lower than those needed for low-LET radiation.     

Ganglioside Composition of Normal and Mutant Mouse Embryos

The enrichment of gangliosides in neuronal membranes suggests that they play an important role in CNS development. We recently found a marked tetrasialoganglioside deficiency in twl/twl mutant mouse embryos at embryonic day (E)-11. The recessive twl/twl mutants die at embryonic ages E-9 to E-18 from failed neural differentiation in the ventral portion of the neural tube. In the present study, we examined the composition and distribution of gangliosides in twl/twl mutant mouse embryos at E-12. The total ganglioside sialic acid concentration was significantly lower in the mutants than in normal (+/-) embryos. The mutants also expressed significant deficiencies of gangliosides in the "b" metabolic pathway (GD3, GD1b, GT1b, and GQ1b) and elevations in levels of gangliosides in the "a" metabolic pathway (GM3, GM2, GM1, and GD1a). These findings suggest that the mutants have a partial deficiency in the activity of a specific sialyltransferase in the b pathway. Regional ganglioside distribution was also studied in E-12 normal mouse embryos. The ganglioside composition in heads and bodies was similar to each other and to whole embryos. Total ganglioside concentration and the distribution of b pathway gangliosides were significantly higher in neural tube regions than in nonneural tube regions. These findings suggest that b pathway gangliosides accumulate in differentiating neural cells and that the deficiency of these gangliosides in the twl/twl mutants is closely associated with failed neural differentiation.     

Microbodies in fungi: a review

Summary Microbodies are ubiquitous organelles in fungal cells, occurring in both vegetative hyphae and spores. They are bounded by a single membrane and may contain a crystalloid inclusion with subunits spaced at regular intervals. Typically, they contain catalase which reacts with the cytochemical stain 3,3-diaminobenzidine to yield an electron-opaque product, urate oxidase,l--hydroxy acid oxidase andd-amino acid oxidase. Their fragility and the necessity to disrupt the tough fungal cell wall before isolating them make them difficult to isolate. Analysis of enzymes in purified or partially purified microbodies from fungi indicates that they participate in fatty acid degradation, the glyoxylate cycle, purine metabolism, methanol oxidation, assimilation of nitrogenous compounds, amine metabolism and oxalate synthesis. In organisms where microbodies are known to contain enzymes of the glyoxylate cycle, they are known as glyoxysomes; where they are known to contain peroxidatic activity, they are known as peroxisomes. In some cases microbodies contain enzymes for only a portion of a pathway or cycle. Thus, they must be involved in metabolic cooperation with other organelles, particularly mitochondria. The number, size and shape of microbodies in cells, their buoyant density and their enzyme contents may vary with the composition of the medium; their proliferation in cells is regulated by the growth environment. The isolation from the same organism of microbodies with different buoyant densities and different enzymes suggests strongly that more than one type of microbody can be formed by fungi.     

Arabidopsis: Sensitivity of growth to high temperature

Arabidopsis thaliana seedlings as measured by an electrolyte leakage assay, have been found to be extremely sensitive to high temperature stress as compared to a high temperature tolerant variety (Tracy) of soybean. Over 50% ion leakage occurred in Arabidopsis leaves during a 15-minute exposure to 50°C, indicating a heat killing time of less than 15 minutes. In contrast, the heat killing time for soybean at 50°C was over five times longer. When soybean or Arabidopsis seedlings in culture plates were exposed to 37°C for 2 hours and then returned to 23°C, they suffered no apparent short-term or long-term damage. Soybean seedlings given a 42°C, treatment for 2 hours also showed no damage. Arabidopsis seedlings after a 42°C treatment for 2 hours showed no apparent immediate damage, but 48 hours after return to 23°C severe damage symptoms were visible and after 96 hours all the seedlings were dead. Both soybean and Arabidopsis seedlings synthesize heat shock proteins (hsps) when exposed to 42°C for 2 hours. The hsps synthesized are of similar molecular weights, although the relative abundances of the different size classes are very different in the two plants. Even though hsps are produced in Arabidopsis seedlings after a 2 hour exposure to 42°C their presence is not sufficient for the seedlings to recover from the effects of rhe heat shock when returned to 23°C. Our results show that Arabidopsis has a heat sensitive genotype. This along with its other characteristics should make it a good model system in which to assay in transgenic plants, the functions of homologous and heterologous genes that might be candidates for determining heat tolerance in plants.